Cancer is a disease of monumental proportion and a leading cause of death in the world population. Of the estimated 1,130,000 people in the United States diagnosed in 1992 as having cancer (excluding skin cancer), about half of them will die from these diseases. Smart, et al., "Cancer Screening and Early Detection," Cancer Medicine, (Chapter VII, p. 408, Holland, et al. editors, 1993).
In response to this cancer threat, the medical community has sought the development and employment of a number of tools for screening and early detection of neoplasms (i.e., benign or malignant tumors) to facilitate early treatment, especially when there is a high cure rate. In other words, it has long been a goal to determine whether a person has a cancer before the person becomes symptomatic (and is thus asymptomatic) or the cancer has grown to the point of becoming advanced, difficult to treat and with a poor prognosis.
Likewise, after a patient has become symptomatic of cancer, screening follows. However, screening tests are not always conclusive of the existence and type of cancer besetting the patient. Thus, typically a diagnosis is preceded by a regime of various screening and diagnostic tools (including, e.g., physical examinations, exploratory surgery, medical imaging, assays, and the like). The results of the regime are evaluated by the physician or team, and a diagnosis is formulated. There is, quite unexpectedly, no limit to diagnostic tools providing overlapping information, since confirmation of a condition, prognosis and treatment are all critical. Certain of these tools are less intrusive than others. Thus, serum assays (and assays of other biological samples) play an important role in the diagnostic process of a patient that has become symptomatic in better determining the type, nature and progress of the disease.
Lastly, where a patient with a carcinoma has received therapy (e.g., surgery, chemotherapy, radiation therapy, hyperthermia, and the like), in certain instances it is important to monitor the progress of the post-therapeutic patient to determine the status or recurrence of the cancer. Accordingly, screening methods are employed to, among other things, assay (i.e., qualitatively and/or quantitatively test for cancer indicators) serum of such patients to detect, as early as possible, indicators of exacerbated or relapsing disease after primary treatment is given, or even proper response to such treatment (e.g., chemotherapies).
The carcinomas for which early detection would potentially improve diagnosis include ovarian cancer, breast cancer, prostatic cancer, and pancreatic cancer. Ovarian carcinoma, common in Western countries, has a high mortality rate and thus is of great interest and concern to medical science and particularly to gynecology and medical endocrinology. Ovarian cancer is usually diagnosed at a late stage when it is widespread and after the disease has presented itself clinically. This situation is due primarily to the inaccessibility of the ovaries to physical examination and because early localized cancer has no symptoms. Thus, as a tumor mass starts to grow it is virtually transparent to the patient and does not cause pain or symptomatically interfere with other organs. Screens for ovarian cancer include transvaginal ultrasound for high risk cases, serum markers, transpelvic ultrasound and physical examination. Certain of these examinations are generally too expensive for screening of random populations (specifically ultrasound), others are generally unreliable as a screen (physical examination), and still others offer false positives for ovarian cancer (like most ultrasonic methods).
Prediagnostic tests include imaging (X-ray, ultrasound, MRI), and exploratory surgery (laparoscopy and laparotomy). When the results are assembled, a tentative diagnosis ensues, which must be confirmed by histological analysis. Any screening test that allows improved sensitivity and selectivity renders the test reliable for large populations and possesses obvious utility for cancers in general, and ovarian cancer in particular. Since cancer is typically clinically diagnosed after it is apparent, a screening test that allows an earlier diagnosis and a proper therapeutic regime to prevent exacerbation of the condition and an early remission are very desirable goals for this disease.
Similarly, breast cancer is often not detected until a palpable mass is discovered either by a patient's own physical examination or during a physician's physical examination. Further, while mammography is presently employed in screening for breast cancer, it is not without risk, e.g., radiation-induced malignancies, has a relatively high cost especially since a trained radiologist or team must evaluate each film, and is of limited value in pre-menopausal women. Therefore, a screening test for determining the existence of breast cancer indicators prior to the presentation of a lump has obvious utility, and can provide a basis for early diagnosis and non-surgical treatment regimes.
Also, prostatic cancer is the most frequently diagnosed cancer in men in the United States (after skin cancers), second only to lung cancer in Canada, and thus is of great concern. A difficulty encountered in screening for prostatic cancer involves benign prostatic hypertrophy, a condition which causes enlargement of the prostate gland and often is accompanied by urethral constriction. Therefore, physical examination by a doctor and/or diagnosis based on symptomatology is inadequate due to the low selectivity of the physical examination and the presentment of symptoms as an indicator of malignancy. There is at least one known blood test for prostate-specific antigen ("PSA") which is presently employed for screening. However, PSA has equivalent sensitivity as digital rectal examinations ("DRE"), and thus sensitivity for early diagnosis of prostate cancer is low. Accordingly, any screening test to increase sensitivity and early diagnosis of prostate cancer has obvious utility and is a sought after goal of the medical community.
Pancreatic cancer is amongst the worst known cancers in man, since presentation is usually followed by an uncomfortable and rapid death. There is no medically recognized screen for pancreatic cancer, nor any treatment other than palliative measures and pain relief. Accordingly, without doubt, an effective screen that in any sense improves the ability of a physician to detect pancreatic cancer at an early stage, may allow for effective treatment in a situation presently lacking any hope for cure by identifying the disease at an early stage.
As a consequence of the metabolic derangement associated with malignancy, malignant cells often inappropriately produce proteins and other substances that, while having no recognized biological value under the circumstances, nonetheless are associated with the particular malignancy (called "tumor-specific markers"). Thus, it is extremely helpful to have a test that searches for the presence of such tumor specific markers, then compares the quantities found against a base line from the patient and/or from a statistical data base, and thereby allows a determination of the probability of the existence of the tumor. The goal of such a screening test is high sensitivity such that no diseased patient remains undiagnosed. However, if the selectivity is insufficient, the screen becomes inappropriate for general patient population use, since the number of false positives is unacceptably high. By definition, a false positive is only detected after further diagnostic follow-up, which may be both costly and inconvenient as well as emotionally traumatic to the patient. It remains a well recognized goal of any medical screening program to reliably diagnose a condition or disease, while minimizing the cost to society and burden to the patient, through techniques that are both sensitive and selective.
Existing serum screens for cancers normally seek to detect the presence of a tumor-specific antigen by the use of an antibody-based assay. Thus, the antibody seeks to bind with an antigenic determinant (usually a portion of the tumor specific marker) which is instrumental in the detection procedure. "Assays" are processes for measuring the serum level of antigenic determinant, which, when outside the defined normal boundaries, are suggestive of disease. Assays and screens are by definition well-known to the art. However, there is always a need to create new or improved specific chemical, biochemical, or immunochemical compositions, procedures, kits and the like, or combinations thereof, which assist in determining serum levels of specific tumor markers. Since it is believed possible to produce and/or provide antibodies that are each specific for different antigenic determinants, and since a number of different antigens may be present in the biological sample, there is also room to test a single sample for the presence of a plurality of antigens. Accordingly, it is known in the art to create a kit that allows for testing a single serum sample for the presence of a plurality of serum antigens. This kit can be used for determining the presence of a number of different diseases, or for simultaneous detection of a number of markers indicative of the same or other diseases in order to improve the sensitivity and selectivity of the procedure.
It is known that hormonally responsive cells can be stimulated using hormone receptor agonists. It is also known that certain tumors are hormonally responsive, because such tumors possess receptors and the mechanisms for responding to such receptor stimulation. As a consequence of the abnormality of tumor cells, response by such cells to hormone stimulation may be aberrant. Therefore, the art has suggested specific examples, in which a response to a hormone stimulation test through the release of a significantly increased amount of a predetermined substance may be diagnostic for the condition. In other words, in a positive result for a stimulation test, an agonist is administered and the agonist causes the tumor cell to release a significantly increased amount of the predetermined substance, and this increased amount is quantitized and, if it is above a certain limit, is presumptive of the existence of the tumor.
Hormonally responsive cells release substances when receptors for the hormone bind the hormone. Likewise, certain analogs of such hormones, i.e., chemical variants of the hormone, are known to bind to the same receptor as the hormone itself, although the effects (agonize, antagonize, or other) may be unknown.
It is known in the art that GnRH agonist compounds, when chronically administered to patients having ovarian cancer, may over the long term result in a suppression of the disease and of tumor specific markers such as CA 125. Savino et al, "GnRH Analogs in Gynecological Oncology: A Review", J. Chemotherapy, 4(5):312-320 (1992). Thus, the art teaches that the administration of GnRH decreases the levels of tumor markers, even though it is known that GnRH analogs may stimulate the ovaries. Parinaud et al., "Paradoxical Ovarian Stimulations in the use of LHRH Analogs", Eur. J. Obstet. Gynecol. Repro. Biol., 47:129-133 (1992). The art suggests that, with respect to human endometrial cell lines, GnRH analogs exert an antiproliferative effect measurable within the first 24 hours. Emons et al., "High Affinity Binding and Direct Antiproliferative Effects of Luteinizing Hormone-Releasing Hormone Analogs in Human Endometrial Cancer Cell Lines", J. Clin. Endo. Metab., 77(6):1458-1464 (1993).
Serum ovarian cancer markers have not been as valuable as originally hoped for by the medical community. In standard tests, false positives occur with some frequency, especially in menstruating women with endometriosis or pelvic inflammatory disease. Since these conditions are relatively common and do not, by themselves, exclude a diagnosis of ovarian cancer, the usefulness of this test as a screen for the general population is limited. Moreover, positive results in a CA 125 test may also occur in other gynecologic malignancies and nongynecologic malignancies. Therefore, a positive result does not tell a treating physician if there is a tumor, or if present where it is or what type it is.
Standard tests are also limited in their usefulness by the occurrence of false negatives. Employing the standard testing method, up to 50% of early ovarian cancer may be unassociated with a rise in serum CA 125. This result is particularly unacceptable because a local stage I ovarian cancer has a 90% cure rate while an advanced ovarian cancer is associated with only a 35% five year survival rate.
It is known in the art to administer a compound to stimulate certain endocrine-tissue derived tumors, wherein response to the stimulation is measured by increased serum quantities of endocrine products of the tumor, which serve as specific markers, during a short period of time after administration of the compound. For example, it is known to administer calcium or pentagastrin to determine the presence of a possible thyroid medullary carcinoma, measuring serum calcitonin before and after administration, the resulting calcitonin level after stimulation being markedly increased. Barbot et al., "Pentagastrin Stimulation Test and Early Diagnosis of medullary Thyroid Carcinoma Using an Immunoradiometric Assay of Calcitonin: Comparison with Genetic Screening in Hereditary Medullary Thyroid Carcinoma ", J, Clin. Endocrinology and Metabolism (1994). Likewise, catecholamine release can be stimulated from pheochromocytoma (tumor of the adrenal medulla) by the administration of glucagon. Both of these tumors are believed to be primary endocrine neoplasms and may be related to each other and to parathyroid adenomas, in a familial (autosomal dominant) or a sporadic pattern. These tumors may also be related to neuroectodermal syndromes. Williams, Textbook of Endocrinology, W. B. Saunders, Philadelphia (1974) 306-316, 764-67, 1022-23.
The art does not teach stimulatory tests in general to screen for neoplasms. See, Deligdisch, Altchek & Cohen, Atlas of Ovarian Tumors, Igaku-Shoin, New York (1994)(Prepublication, a copy being provided as an appendix), incorporated herein in its entirety by reference. Accordingly there are no teachings of a screen for neoplasms using stimulatory tests in general, and especially with respect to ovarian, breast, prostatic, or pancreatic cancers.
With respect to ovarian cells, it is heretofore unknown that release of serum tumor marker can be differentially stimulated by administering hormone analogs, thus increasing the utility of such analogs in screening for tumors by distinguishing over non-ovarian sources of such markers. In particular, GnRH receptors are present on ovarian epithelial neoplasms as well as on normal ovarian tissue. Emons et al., "Gonadotrophin Releasing Hormone Binding Sites in Human Epithelial Ovarian Carcinomata ", pp. 215-21; Ohno et al., "Presence of Gonadotrophin-Releasing Hormone and its Messenger Ribonucleic Acid in Human Ovarian Epithelial Carcinoma", Am J. Obstet. Gynecol., 169:605-10 (1993); Adelson et al., "Effects of Gonadotropin-Releasing Hormone Analogs on Ovarian Epithelial Tumors", Clin. Obstet. Gynecol., 36(3):690-700 (1993); Leung et al., "Intracellular Signalling in the Gonads", Endocrine Reviews, 13(3):476-498 (1992)(incorporated in its entirety by reference). Long term treatment of patients harboring these neoplasms with GnRH analogs has been associated over time with a decline in the tumor marker CA 125 thus suggesting a possible desensitization effect.
CA 125 is a high molecular weight, non-mucin cell surface glycoprotein complex of molecules that is present in forms varying from 220 to over 1000 kD. CA 125 is identified by a murine monoclonal antibody called OC 125. CA 125 has a serum half life of 4.5 days, and has an upper normal range of 35 U/ml.
The CA 125 antigen complex is normally found in cells from the coelomic epithelium, and may also be found in the epithelia of the pancreas, colon, gallbladder, stomach, lung and kidney. Various benign conditions may cause elevated CA 125 levels, including endometriosis, adenomyosis, leiomyoma, acute pelvic inflammatory disease, early pregnancy, menstruation and benign ovarian cysts.
The selectivity of CA 125 as a screen for cancer is limited, being 97% for a single serum assay, and the art has sought to improve screens employing serial testing, combinations of serum markers, and by adding pelvic examination and ultrasound. The sensitivity of CA 125 as a screen is only about 53%, which, of course suggests a large number of false negative results, which occur especially in stage I tumors. It is these early, stage I tumors which are desired to be detected because of their far improved prognosis. Therefore, CA 125 serum testing, as presently performed, is an inherently unreliable screen, with a low selectivity and a low sensitivity, and therefore is insufficient as presently devised.
The effects of the selectivity of a screen are apparent from the following analysis. Ovarian cancer has an incidence of 40 per 100,000 women over age 45 per year. Therefore, in order to achieve a 100% sensitivity, e.g. detection of all diseased patients, with at least 10% positive predictive value, e.g. no more than 9 false positives for each detected tumor, a 99.6% specificity value would be required. This is in contrast to the actual about 97% selectivity and about 50% sensitivity. Therefore, CA 125 remains experimental as a screen for ovarian cancer, missing many early tumors and requiring a follow-up of a large number of false positive results. However, CA 125 may have significant value in following post-treatment ovarian cancer, where a serum measurement is compared to a baseline measurement, thereby facilitating monitoring CA 125 levels as an indication of tumor mass, providing evidence of the efficacy of chemotherapy and providing an early indication of relapse. See Berkowitz, "CA125 Measurement in the Epithelial Ovarian Cancer: a 10-Year Anniversary of Clinical Investigation", Gynecol. Onc., 49:1-2 (1993).
Other proposed serum indicators of ovarian tumors include inhibin activin, follistatin, Lipid-Associated Sialic Acid (LASA-P), CA 19-9, CEA, MB-70K, DM/70K (Dianon Systems, Inc. Stratford, Conn.), M-CSF, Urinary Gonadotrophin Factor, Ca 130 and PRL. See Bernstein et al., "Comparison of CA 125, Lipid Associated Sialic Acid (LASA-P), CA 19-9, and CEA in Monitoring Patients with Ovarian Cancer", J. Tumor Marker Oncology, 6(3):183-187 (1991); Miyanaga et al, "Differential Control of Activin, Inhibin and Follistatin Proteins in Cultured Rat Granulosa Cells", Biochem. Biophys. Res. Comm. 194(1): 253-258 (1993); Schwartz et al, "Circulating Tumor Markers in the Monitoring of Gynecologic Malignancies", Cancer, 60(3):353-361 (1987); Cole et al., "Urinary Gonadotropin Fragment, a New Tumor Marker", Gynecol. Onc. 36:391-94 (1990); Suzuki et al., "Macrophage Colony-Stimulating Factor as a Tumor Marker for Epithelial Ovarian Cancer", Obstet. Gynecol., 82(6):946-950 (1993)(incorporated herein in its entirety by reference); Hosono et al., "Different Antigenic Nature in Apparently Healthy Women with High Serum CA 125 Levels Compared with Typical Patients with Ovarian Cancer", Cancer, 70(12):2851-2856 (1992). Other known markers include CA 15-3, CA 72-4, CA 54/61 and OVX-1. Negishi et al, "Serum and Tissue Measurements of CA72-4 in Ovarian Cancer Patients", Gynecol. Onc., 48:148-54 (1993); Kobayashi et al, "Monoclonal Antibodies MA54 and MA61 as Potential Reagents in the Prognosis of Patients with Ovarian Cancer", Gynecol. Onc., 49:80-85 (1993); Woolas et al, "Combinations of Multiple Serum Markers are Superior to Individual Assays for Discriminating Malignant from Benign Pelvic Masses ", Preprint (incorporated herein in its entirety).
Inhibin, a naturally occurring peptide hormone is known to increase in quantities which, according to one study peak at 772.+-.38 U per liter in the follicular phase of the menstrual cycle in pre-menopausal women, and is undetectable in most post-menopausal women. Lapp ohn et al, "Inhibin as a Marker for Granulosa-Cell Tumors," New England Journal of Medicine, Vol. 312, No. 12, Sept. 21, 1989, pp. 790-793. See also Kauppila et al., "GnRH Agonist Analog Therapy in Advanced/Recurrent Granulosa Cell Tumors: Further Evidence of a Role of Inhibin in Monitoring Response to Treatment", Gynecological Endocrinology, pp. 271-274. Likewise, inhibin is believed to increase in serum level concentrations for patients with granulosa-cell tumors. Healy, et al., "Elevated Serum Inhibin Concentrations in Postmenopausal Women with Ovarian Tumors," New England Journal of Medicine, 329(21): 1539-42 (Nov. 18, 1993); Witt et al, "Endocrine Function of Granulosa Cell Tumors In Vivo", Gynecol. Obstet. Invest., 33:59-64 (1992). Serum inhibin levels may be quantified according to the method of Medgenix Diagnostics, Fleurus, Belgium, described in Blaakaer et al., "Immunoreactive Inhibin-Production in Post-Menopausal Women With Malignant Epithelial Ovarian Tumors", Eur. J. Ob. Gyn. and Reproductive Biol., 52 (1993) 105-110, incorporated herein by reference.
Hormones are generally considered to be circulating compounds which produce a specific biological effect on responsive cells. Hormones are detected by these responsive cells by means of receptors, which are generally on the cell surface or in the cytoplasm. Peptide hormones generally are bound to cell surface receptors, producing a secondary messenger signal, such as c-AMP, in the cell. Often, circulating hormones have a short half-life, since they are degraded, inactivated or removed from the circulation. In order to provide improved pharmacological properties of a drug which acts similarly to a natural hormone, modifications may be made to increase its binding to the receptor or increase its half-life. Compounds which closely resemble natural hormones with modifications and/or substitutions and having biological activity as receptor agonists or antagonists are known as hormone analogs. These analogs may be modified by replacing certain 1-amino acids with d-amino acid isomers or by derivatization. Hormone analogs also include amino acid substitutions and peptides having shorter or longer chain lengths.
While the endocrine system generally requires the transmission of a chemical message through the blood stream to a distant target organ, a paracrine signaling system also exists which allows chemical signaling between proximate cells, without requiring transmission through the blood stream. In many cases, endocrine hormones may also serve as paracrine hormones having a different function. Thus, the same or similar endocrine hormones having a defined effect on an endocrine target organ may also have unexpected paracrine or autocrine effects on various organs, if the hormone is functionally present in the intercellular space in sufficient quantities.
Large numbers of peptide hormone analogs are known, and the selection of a particular analog for biological or medicinal use is within the ordinary skill in the art once the receptor organ and the desired effect are identified. For example, in order to maximally stimulate tissues which are known to be responsive to GnRH (gonadotrophin releasing hormone), one skilled in the art would select a protease resistant GnRH receptor agonist. In particular, such hormone analogs are FDA approved, e.,g., Synarel and Nafarelin, and can be administered to patients. There are other examples of FDA approved peptide hormones, and a host of experimental and non-U.S. drugs available.
It is therefore an object of the present invention to provide a dosage form, method of administration, assay, screen, and kit for the general detection of neoplasms.
It is another object of the present invention to provide a dosage form, method of administration, assay, screen and kit for the detection of specific neoplasms, namely ovarian cancer, breast cancer, prostatic cancer, and pancreatic cancer.
Other and further objects of the invention will become apparent through a reading of the Summary, Detailed Embodiments and Claims, below.